Recording scanner for chromatograms having radio activity



March 27, 1962 E. B. FOWLER ETAL 3,027,458

RECORDING SCANNER FOR CHROMATOGRAMS HAVING RADIO ACTIVITY Filed Aug. 19,1955 2 Sheets-Sheet 1 INVENTORS ATTORNEY RECORDING SCANNER FORCHROMATOGRAMS HAVING RADIO ACTIVITY Filed Aug. 19, 1955 March 27, 1962E. B. FOWLER ETAL 2 Sheets-Sheet 2 f5 I I I 4%; ATTORIVFY 3,d27,d58REQORDKNG SQANNLER FOR CHRQIVEATGGRAh/ih HAVNG RADEO ACTEVHTY Eric B.Fowler and Wayne A. Rhiuehart, Amos, Iowa, assignors to town StateCottage Research Foundation, Inc, Ar'njes, Iowa, a corporation of IowaFiled Aug. 19, W55, Ser. No. 529,415 Ciaims. (C 250--83.6)

This invention relates to apparatus for surveying chromatograms havingradio activity associated therewith, and more particularly to automaticrecording scanner apparatus useful in scanning chromatogramized strips,detecting any radio activity associated therewith and recording theactivity as to extent and location.

Chromatographic techniques have been developed in recent years for theseparation of components of heterogeneous mixtures. Separation isaccomplished generally by placing such a mixture on a supporting medium,such as a paper strip, and immersing one end of the medium in amulti-phasic solution so that the solution ascends or descends along themedium. Components of the unknown mixture will travel along the mediumstrip at difierent rates and thus be separated. The components may beidentified later by spraying the strip with an indicator solution. Sincemicro amounts of each component may be detected in this Way, thetechnique is valuable for the identification of products in an enzymaticreaction mixture wherein the concentration of the components isunusually low.

Radio tracer techniques have also been recently developed and applied tothe field of biology. The radio tracer method is now used extensively todemonstrate the up-take or incorporation of many compounds by biologicalsystems. Tracer techniques may also be used to prove or disprove certainhypothesized chemical reac tions. The uptake of C 0 and itsincorporation into amino acids, and hence protein of cellular systems,may be cited as an instance.

The combination of chromatographic and radio tracer techniques places inthe hands of the research worker a valuable tool for the detection ofproducts of reactions and their relationship to know precursors. Bythese two methods, micro amounts of a product can be separated at a highlevel of radioactivity or a high degree of purity, and the incorporationof labeled fractions demonstrated. However, the two techniques have beenlimited in application in that the element used extensively as a tracer(C is difiicult to detect by conventional means because of the lowenergy of its radiations. The flow type detector tubes available whichwere capable of detecting low activity were of such construction thatthe chromatographs had to be cut into small sectionsand the sectionssurveyed individually. Such a process is wasteful of both time andmaterial. Isolated biological systems in general do not incorporate highconcentrations of reactants, and :since the activity associated with anyone zone might appear in several sections, a dependable total countcould not be obtained.

It is, accordingly, an object of this invention to provide :a means forovercoming the limitations set fonh above, and to thereby extend theapplicability of radio-tracer and chromatographic techniques. Anotherobject of the invention is to provide apparatus designed to solve manyof the limitations heretofore present in the art and to provide aresearch Worker with a dependable tool operative to increase theaccuracy of his results, decrease the time required for any onedetermination, and to be of such an automatic nature as to require noattention after its being set in motion, thereby freeing the worker for.other endeavors.

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Still another object of the invention is in providing apparatus forscanning and recording indicia provided by chromatograms having radioactivity in which the chromatograms are enclosed in a gas-tight housingwhich is an integral part of the counting chamber, whereby no windows orother barriers to the passage of the low energy radiations of the tracerelement into the counting mixture are present. Yet another object is inthe provision of scanning means for chromatograms comprising a definingslot interposed between the chromatogram strip and the actual countingportion of the tube, whereby the area of the strip being surveyed at anyinstant is determined by the size of the defining slotthe apparatusbeing equipped with means for readily altering the size of the definingslot so that the resolution of the apparatus may be varied.

Another object of the invention is in the provision of a drum rotatableand movable axially within a chamber provided by a casing thereabout,the drum being adapted to carry chromatogram strips wound spirally alongthe cylindrical surface thereof, whereby the strip is progressivelyscanned by a radio activity detector positioned in proximity with thedrum and a strip carried thereon, and preferably separated therefrom bya plate having a resolution slot therein aligned with the detectorelement. Still a further object is in the provision of scanning andrecording apparatus of the character described whereby the movement ofthe chart of the recorder is synchronized with the movement of thechromatogram strip in its scanning relation with the detector element sothat following a scanning operation the recording chart and thechromatogram strip can be placed side by side and the areas of radioactivity accurately located. in such an arrangement the relative amountsof activity in the various areas can be determined by integrating thearea of each locatirig peak on the recorder chart.

Yet a further object is that of providing scanning apparatus asdescribed wherein chromatogram strips are carried on a drum mountedwithin a substantially closed chamber, means'being provided in thesystem for evacuating and flushing the chamber and for filling the samewith a counting gas (.a commercially available counting gas known as Qgas may be used), and the pressure in the chamber accurately maintainedat the same predetermined value. Additional objects and advantages willbecome apparent as the specification develops.

An embodiment of the invention is illustrated in the accompany drawing,in Which- FIGURE 1 is a front view in elevation of the scanning andrecording apparatus; FIGURE 2 is a side' view in elevation of theapparatus; FIGURE 3 is a longitudinal sectional view or the sample boxtaken on the line 3 3 of FIGURE 2; FIGURE 4 is a transverse sectionalview of the sample box taken on the line 4-4 of FIGURE 1; and FEGURE 5'is a top plan view of a resolution plate used in the apparatus, andwhich is seen most clearly in FIGURE 3.

The advance provided by the instant invention may be better appreciatedwhen viewed with the background of the prior art in mind. Prior to thedevelopment of the instant invention, several methods were attempted tocircumvent the limitations set out hereinbefore concerning thechromatographic and radio tracer techniques. One method entailed placingthe chromatogram on sensitive film and obtaining a radio autograph whichlocated the positions of the radio active areas. However, highconcentrations of activity were required for successful use of thistechnique, and the method lacks accuracy when total activity associatedwith an active spot is required. Further, the time required for filmexposure is relatively long, and the results of an experiment are notimmediately available.

A second technique involves the use of replicate chromatogrammed strips.One strip is sectioned, and each section counted or detected separately.If the sections are counted in a flow type counter, low activities arenot a primary problem since in the flow counter the sample is placed inthe tube, and there is no loss of counts due. to window absorption as inthe end window tube. Disadvantages of this technique lie in the factthat a large number of small sections must be surveyed which requirescontinued supervision of the counting mechanism over long periods andthe use of large volumes of counting gas for the flow tube. For example,a 2" by 20" chromatogram strip will yield 160 half inch circular discs.Each disc must be mounted separately which means that the flow tube mustbe opened, samples changed, the tube out-gassed or flushed, and thesample counted. Assuming typical counting rates, each operation wouldrequire about thirty minutes, or a total of about 80 hours per 20 strip.Even so, the worker has only a poor estimate of the total activity atany one position of the strip, and the strip is useless for furtherstudy.

With the technique and apparatus herein being considered, the operationis simple and the results are highly accurate. Several strips may besurveyed or scanned with one loading, and constant supervision of theinstrument is not necessary once selection of the type of survey hasbeen made and the apparatus set in operation. A minimum amount ofcounting gas is needed for the survey since the system is sealed and gasdoes not flow through the tube during operation of the apparatus. surveymay be made in about seven and one-half hours automatically, as comparedto over 400 hours that would be required for a comparable survey in thedisc-flow counter method. In addition, the total activity associatedwith the strip spot is read directly from the recorder chart, and thestrip remains intact for further study. The position of activity isindicated on the recorder chart as well as total activity; lowactivities are detected, and a permanent record of the results isobtained.

In referring to the drawings for a detailed description of theapparatus, reference will first be made to FIGURES 1 and 2 showing thescanning and recording apparatus in its entirety. The apparatus isdesignated generally with the numeral 10, and for portability is carriedon a base or platform 11 equipped with castors 12 and a verticallyextending frame 13 having a top wall 14 and intermediate shelves 15 and16. Carried on the shelf 15 is a rate meter assembly 17 comprised ofelectronic circuits and components and, as is seen in FIGURE 1, isprovided with a front panel 18 having a volt meter 19 and micro ammeter20. Mounted on the shelf 16 is a recorder 21 that may be a Brownrecorder and is equipped with a front panel 22 (FIGURE 1) having acentral window 23 therein that provides visibility for a recording chartadvanced by the apparatus of the recorder.

Mounted on the platform 11, as is shown in FIGURE 2, is a tank 24adapted to contain under pressure a volume of an inert counting gas. Thetank is equipped with a manually operable valve 25 that leads into aconduit 26 that carries the gas into the chamber of a sample box 27 thatwill be described in detail hereinafter. That platform 11 also supportsa motor driven pump 28 that is a high vacuum pump and, if desired, maybe a high vacuum Cenco pump. The pump is connected to a manifold 29 thatopens into a manifold extension 30 equipped with a Sylphon high vacuumshut-off valve 31. The extension 30 communicates with the sample box atfour spaced apart points through branch conduits designated by thenumerals 32a through 32d. The frame 13 also carries a mercury manometer33 having a meter stick 34. The manometer is connected to the manifoldbranch 30 through a valve 35 and is employed to measure the pressure ofthe counting gas within the sample box 27. The sample box 27, referringnow to FIGURES 3 and 4, comprises a generally cylindrical casing36providing a chamber 37 therein and a drum 38 mounted within A double thechamber 37. The casing 36 at one end thereof is equipped with an endclosure wall 39 that may be squareshaped, as seen in FIGURE 2, anddimensionally is larger than the casing 36 so that it provides outwardlyextending flanges that may be employed in mounting the casing on the topwall 14 of the frame. At its opposite end the casing 36 is equipped withan outwardly extending perimetric flange 4t) equipped with a pluralityof spaced apart threaded openings therein that are adapted to receivethreaded screws 41 that may be provided with wing nut heads 42, whichare employed in securing the end wall 43 to the flange 40. As is shownin FIGURE 3, the end wall 53 is provided at a spaced distance inwardlyfrom the edge thereof and along its inner face with an annular channel44 having seated therein a sealing gasket 45. The end walls 39 and 43 incombination with the casing 36 provide a substantially fluid-tightchamber 37.

The drum 3% comprises a cylindrical shell or tube 46 that is hollow andthat is provided with spaced apart wall members 47 and 48, each of whichis equipped with a plurality of central openings 49 therethrough andperipherally located openings 50. The walls 47 and 43 have alignedcentral openings provided with keyways therein and seat upon a shaft 51equipped with a key 52 that is journalled for rotation adjacent the endwall 39 in a bearing 53 secured to the end wall by cap screws 54-, andin a bearing 55 carried exteriorly of the end wall 43 and securedthereto by screws 56.

Referring now to FIGURES l and 3, it is seen that the shaft 51 extendsoutwardly from the end Wall 43 and is connected through a gear reducer57 to a synchronous motor 58. When the motor is energized, rotation ofthe shaft 51 rotates the drum 33 which is locked thereon to preventrelative rotational movement therebetween but is free to move axiallyalong the shaft. By means of appropriate gear ratios in the gear reducer57, the rate of travel of the periphery of drum 38 may be made the sameas the rate of travel of the recording chart on the Brown recorder, sothat the movement of the chart of the recorder is synchronized with themovement of the drum 38 within the chamber 3'7.

The tube or shell 46 of the drum 38 is provided about thecircumferential outer surface thereof with a spiral channel or groove 59which extends from end to end thereof. Adapted to ride in the spiralgroove 59 is a guide pin 60 that extends through a boss 61 with whichthe casing 36 is provided. The boss 61 is threaded at its outer end andthreadedly receives a cap 62 that bears downwardly on a flange 63carried by the guide pin to force the same into tight engagement with aresilient seal member 64, whereby the mounting for the guide pinprovides a substantially fluid-tight seal preventing the escape of gasesfrom the chamber 37. It is clear from FIGURE 4 that the portion of thepin 60 that rides in the groove 59 may be reduced in cross section. Itwill be appreciated that the rotation of the drum 38 will cause the drumto be moved axially or bodily along the shaft 51 because of theengagement between the guide pin and the spiral groove.

As is shown most clearly in FIGURE 3, the casing 36 is provided alongthe wall thereof intermediate its ends with an opening 65 and with ashort tubular conduit 66 that extends outwardly from the casingthereabout. The tube 66 is provided with a laterally extending flange 67that has an annular channel in the upper face thereof that receives agasket 68. Seated upon the flange in covering relation with the gasketor seal member is a short tubular section 69 provided with a seal member70 that has seated thereon a laterally extending flange 71 of an outerclosure member 72 provided with a central opening 73 therein. Elongatedbolts 74 secure the flanges 67 and 71 and the member 69 together.Extending outwardly from the closure member 72 about the opening 73therethrough is a collar member 75 that bears against a seal 76 carriedby the closure member 72 to establish a fluidtight relation therewith.Collar member 75 has provided an internal shoulder at its lower openingonto which a Kovar glass seal 75a may be soldered to provide afluidtight relation thereby.

Secured to the collar 75 is a fitting 77 of a coaxial cable 78 that, asis shown in FIGURE 2, is connected to the rate member assembly 17. Thecenter conduit of the cable 78 is electrically connected to a detectorelement or anode 79 that may be a ten mil tungsten Wire provided at itsend with a glass head 80. The detector 79 is aligned with a resolutionslot 81 provided in a plate 82 that seats on an annular flange section83 provided by the casing 36 about the opening 65. The plate 82, as isshown best in FIGURE 5, is provided with a notch 84 that receivestherein a key 85 provided along the inner wall of the member 66. Theresolution slot 81 has a predetermined area, and it will be appreciatedthat the plate 82 is readily removable and may be replaced with anotherhaving a resolution slot of greater or smaller area depending upon thedegree of resolution that may be desired in a scanning operation.

The detector 79 through the slot 81 is adapted to detect the radioactivity of chromatogram strips 86 that are carried by the drum 38 aboutthe outer surface of the tubular shell 46 thereof. The chromatogramstrips are spirally wound about the shell 46 and may extend from end toend thereof. They may be secured in place by a plurality of Phosphorbronze clips 87. In an embodiment of the invention that has beensuccessfully employed, three strips each 2" in width by 20 long may besecured to the shell 46 in end to end relation to provide a continuouschromatogram extending from end to end of the drum. Preferably, the drum38 is formed of brass.

The member 66 is provided with a flow port 88 communicating with theconduit 26 that connects through the valve 25 to the countings gascontainer 24.

A micro switch 89 is carried by the end wall 43 and is provided with apair of leads 90 and 91 that extend outwardly through the wall 43 andhave, respectively, glass seals 92 and 93 thereabout. The end wall 39carries a micro switch 94 having leads 95 and 96 extending therefromoutwardly through the end wall and through glass seals 97 and 98. All ofthe seal members function to provide a fluid-tight closure about thelead wires for the micro switches, and it has been found that Kovarseals may be employed to bond the glass to the metal end walls.

Referring back to FIGURE 1, it is seen that the front panel 101 of theapparatus is equipped with a plurality of switch members designated withthe numerals 102, 103, 104, 105 and 106. Also, pilot lights 107, 108 and109 are provided. The switches respectively are the pump switch forcontrolling the motor of the pump 28, the master switch for controllingthe power connection to the apparatus generally, a momentary switch, aselector switch, and a relay reset switch.

Operation In use of the apparatus, the end wall 43 is released from itsposition of sealing engagement with the flange 40, and the drum 38 ismoved bodily out of the casing 36. The chromatogram strip 86 ispositioned about the shell 46 of the drum and is secured thereto byclips 87. If a plurality of short strips are placed in end to endrelation about the drum, the end portions thereof may be securedtogether by tape or other appropriate means. The drum is then moved intothe casing and is preferably positioned so that the starting end of thestrip 86 is substantially adjacent the resolution slot 81 in the plate82. The end wall 43 is then secured as is shown in FIG- URE 3.

In removing the drum 38 from the casing or returning it to positiontherein, the guide pin 60 may be withdrawn from engagement with thespiral channel 59, or alternatively may be rotated until the guide pinis released from the channel.

When samples or chromatogram strips are changed, the

counting chamber 37 must be flushed and a fresh supply of counting gasunder pressure admitted thereto. In the system provided, it is notnecessary to flush the chamber 37 several times with counting gas priorto starting the run of a new sample. Such a procedure is undesirable forit involves the use of large quantities of gas which is expensive.Instead, the pump 28 is actuated and the chamber 37 is evacuated sincethe pump is connected thereto through the manifold 29, manifoldextension 30 and branch conduits 32a through 32d. After evacuation, asupply of counting gas is admitted to the chamber 37 through the valve25. Preferably, that volume of gas is then evacuated from the chamberand a fresh quantity of counting gas admitted thereto. Such a flushingprocedure substantially eliminates contaminating gas which wouldinterfere with counting tube operation.

To actuate the apparatus, the various switches are moved to the onposit-ion, and the motor 58 through the shaft 51 rotates the drum 38. Asthe drum rotates, the coaction of the guide pin 60 and spiral channel 59causes the drum to move bodily axially within the chamber 37, or towardthe right as viewed in FIGURE 3. The chromatogram strip 86 then advancesprogressively beneath the resolution slot 81 and is scanned by thedetector element or anode 79 of the scanning tube.

At the same time, the recorder 21 is functioning, and it is operativelycoupled to the rate meter assembly 17 which in turn receives thecounting impulses from the detector 79, and the radio activity of thestrip at the location beneath the resolution slot 81 is recordeddirectly on the chart of the recorder. Thus, both the amount or extentof activity and the precise location thereof on the strip 86 aredirectly recorded. This procedure is valuable, for after a test has beenrun the strip 86 may be removed from the drum and placed beside thechart of the recorder so that the extent and exact location of therecorded activity can be transposed to the strip. The extent of activitymay be determined by integrating the areas under the curves or pips onthe recording graph.

The apparatus may be set up so that when the drum 38 engages the switcharm of the micro switch 94, the apparatus will be de-energized. Thus,one pass or one complete scanning cycle of the strip 86 will have takenplace. On the other hand, if it is desired to provide a double-scanningcycle, the selector switch is so positioned, and upon the drum strikingthe switch arm 100, the motor 58 is reversed and the drum will then moveaxially to the left within the chamber 37 until it engages the arm 99 ofthe micro switch 89 which functions to deenergize the apparatus. By asuitable means, the recorder chart is marked at the end of the forwardscan. In such a scanning cycle, strip 86 will be scanned first in onedirection and then in the other.

The plate 82 is removably mounted within the member 66 which permits aplurality of plates 82 to be interchanged with each other. The variousplates will have resolution slots 81 of different size, whereby thedegree of resolution of the scanning operation may be selected by theoperator. Ordinarily, the narrower the slot 81, the greater will be thedegree of resolution. It is also desirable to reduce the operationalspeed of the apparatus where greater resolution is being provided by asmaller resolution slot 81.

The amount of counting gas consumed in operation of the apparatus ismaintained at a minimum because no gas flows through the countingchamber 37 during the testing or scanning of a strip. The only gasmovement through the chamber comes during the flushing operation priorto the scanning of a new test sample. The operation is substantiallyautomatic, and once the apparatus is set in motion and the mode ofoperation selected, attention from an operator is unnecessary for theapparatus scans and records automatically and terminates its ownoperation upon the completion of a scanning cycle, whether the cycle bea single or double-scanning cycle.

While in the foregoing specification an embodiment of the inventionhasbeen ,set forth in substantial detail for purposes of making a fulland complete disclosure of the invention, it will be apparent to thoseskilled in the art that numerous changes may be made in those detailswithout departing from the spirit and principles of the invention.

We' claim:

1. In apparatus for scanning chromatogram strips to detect the radioactivity thereof, a casing providing a counting chamber therein, acounting tube carried by said casing and having a. stationary detectorelement in facing relation with the interior of said counting chamber, adrum having a cylindrical surface for spirally supporting an elongatedand discontinuous chromatogram strip thereon and being mounted withinsaid chamber for rotational and axial movement, and guide meanscooperating with said drum for progressively advancing said drum axiallyas the same is rotated and thereby orienting said spirally woundchromatogram strip, in scanning relation with said stationary detectorelement as said drum is rotated.

2. The apparatus of claim lin which said drum is substantially hollowand is provided with spaced apart partitioned walls each having aplurality of apertures therethrough, and in which a rotatable shaftextending axially through said chamber extends through said walls andprovides a mounting for said drum..

3., In a chromatogram scanning apparatus of the character described, acasing providing a substantially fluid-tight counting chamber therein, arotatable shaft disposed within said chamber and fixed against axialmovement with reference thereto, a. drum carried by said shaft for axialmovement therealong but being constrained on said shaft to preventrelative rotational movement therebetween, said drum having acylindrical outer surface and being provided along said surface with aspiral groove, a stationary guide provided by said casing and adapted toride along 'said groove for progressively and gradually advancing saiddrum axially as the same is rotated, said drum being adapted to supportan elongated spirally-wound and dis continuous chromatogram strip uponsaid cylindrical surface and between the spirals of said groove, acounting tube carried by said casing in open communication with tectorelement and said guide being positioned and arranged so that as saiddrum is axially advanced upon rotation thereof said spirally-Woundchromatogram strip is carried in progressive scanning relation withreference to said detector.

. 4. In an apparatus for scanning chromatogram strips to detect theradioactivity thereof both as to extent and location along said strips,a casing providing a substantially fluid-tight counting chamber, acylindrical drum adapted to carry an elongated discontinuouschromatogram strip in spirally wound fashion along the outer surfacethereof, means for supporting said drum for rotational and axialmovement Within said chamber, a guide element provided by said casingand cooperating with said drum for progressively moving the same alongits rotational axis as said drum is rotated, and a counting tubecommunicating with said chamber and having a stationary detector elementin facing relation with the outer surface of said drum, said detectorand guide elements and said drum being related with respect to eachother so that upon rotational and axial movement of said drum achromatogram spirally wound thereon passes longitudinally in scanningrelation with reference to said detector element, whereby, saidspirally-wound strip is scanned by said detector as said drum is rotatedand axially advanced to detect both the extent and location ofradioactivity therealong.

5. The structure of claim 4 in which said drum is hollow and is providedwith spaced partion Walls each having a plurality of apertures therein.

References Qited in the file of this patent UNITED STATES PATENTS1,806,375 'ifieclernan May 19, 1931 2,490,298 Ghiorso et al. Dec. 6,1949 2,751,505 Anderson June 19, 1956 OTHER REFERENCES Use of Beta-RayDensitometry in Paper Chromatogra- .phy, from Analytical Chemistry, vol.23, No. 1, January 1951, pages 207 to 208.

Apparatus for Automatically Scanning Two-Dimensional Paper Chromatogramsfor Radioactivity, by W. J. Wingo, from Analytical Chemistry, vol. 26,July-December 1954, pp. 1527, 1528.

